This invention relates to methods of gene delivery into cells of the nervous system, for example, cells of the central and peripheral nervous system, by introducing and expressing gene sequences using herpes simplex virus 1 (HSV-1) mutants with deletions in gene(s) for viral replication.
The delivery and expression of heterologous or native genes into cells of the nervous system to alter normal cellular biochemical and physiologic processes in a stable and controllable manner is of substantial value in the fields of medical and biological research. This genetic perturbation of the nervous system provides a means for studying the molecular aspects of neuronal function and offering therapeutic approaches to pathologic processes.
Herpes simplex viruses possess several properties that render them attractive candidates as delivery vehicles to bring foreign genes into cells of the peripheral and central nervous systems. (Breakefield et al., Mol. Neurobiol. 1:339 (1987); Dobson et al., J. Virol. 63:3844-3851 (1989); Ho and Mocarski, Virology 167:279-283 (1988); Ho et al., Proc. Natl. Acad. Sci. USA 86:7596-7600 (1989); Breakefield and DeLuca, New Biologist 3:203-218 (1991); Chiocca et al., New Biologist 2:739-746 (1990); Friedmann, T., Science 244:1275-1280; Palella et al., Gene 80:138 (1989).)
These viruses can infect and deliver their DNA into many different cell types, including adult postmitotic neurons; they can enter a state of latency in neurons including, for example, sensory neurons, in which the viral genome exists as an episomal element in the nucleus of the cell; they are able to infect a substantial number of cells in vivo due to their ability to reach high titers in culture and to propagate in the nervous system and due to the relatively long half life of virus particles; large exogenous DNA sequences can be inserted into their genomes; and they possess a wide host range. (Longnecker et al., In: Viral Vectors, Gluzman et al. (eds.), CSH Lab, pp. 68-72 (1988); Roizmann et al., In: Virology, Fields et al. (eds.), Raven Press, New York, pp. 497-526 (1985); Rock et al., Nature 302:523-525 (1983).)
Recently, an amplicon-type plasmid-vector system based on herpes simplex virus 1 (HSV-1) has been used to achieve relatively stable expression of the lacZ gene in cultured neurons. (Spaete et al., Proc. Natl. Acad. Sci. USA 82:694 (1985); Geller et al., Science 241:1667 (1988); Geller et al., Proc. Natl. Acad. Sci. USA 87:1149 (1990).) In this plasmid vector, the lacZ gene was placed under the control of an HSV-1 immediate-early promoter and packaged using a temperature-sensitive HSV-1 helper virus tsK. Coen et al., Proc. Natl. Acad. Sci. USA 86:4736 (1989); Leib et al., J. Virol. 63:759 (1989); Davison et al., J. Gen. Virol. 65:859 (1984).)
Herpes simplex or other neurotropic viral vectors (Loewy et al., Proc. Soc. Neurosci. 17:603-15 (1991)) offer one potential means of delivering functional genes to alter the physiology of sensory or other neurons. Herpes simplex virus is known to be retrogradely transported from sensory terminals to sensory ganglia by a mechanism of fast axonal transport (Cook et al., Infec. and Immun. 7:272-288 (1973). HSV that reaches the nucleus of a sensory neuron, is capable of either lytic replication or latent infection. Lytic replication is characterized by the production of mature virus particles and destruction of the cell. Latent infection is characterized by the long-term stable presence of viral DNA in the nuclei of infected cells, most likely in the form of an episomal unit (Rock et al., Nature 302:523-525 (1983); Efstathiou et al., J. Virol 57(2):446-455 (1986); Mellerick et al., Virol 158:265-275 (1987)), and transcription that is limited to a specific region of the HSV genome (Croen et al., New Engl. J. Med. 317:1427-1432 (1987); Rock et al., J. Virol. 61:3820-3826 (1987); Spivak et al., J. Virol. 61:3841-3847 (1987); Stevens et al., Science 235:105-1059 (1987); Javier et al., Virol. 166:254-257 (1988); Wagner et al., J. Virol. 62(4):1194-1202 (1988); Kosz-Vnenchak et al., J. Virol. 64:5396-5402 (1990)).
Latent infection with HSV in the absence of lytic replication is possible using replication-defective recombinant vectors mutated by deletion, or insertion, of foreign elements into HSV genes necessary for replication (Dobson et al., Neuron 5:353-360 (1990); Andersen et al., Human Gene Therapy (in press) (1992)). Foreign genes stably expressed by these vectors have included lacZ (Ho and Mocarski ""89) or rabbit xcex2-globin inserted downstream of the putative HSV LAT promoter (Dobson et al., J. Virol 63:3844-3851 (1989)), and lacZ inserted downstream of the Moloney murine leukemia virus long terminal repeat (MoMLV-LTR) retrovirus promoter into the HSV immediate early gene ICP4 (vector 8117/43)(Dobson et al., Neuron 5:353-360 (1990)). Stable expression of foreign genes in the central and peripheral nervous system may also be possible using vectors that are replication-defective due to a mutation in the thymidine kinase (tk) gene. These thymidine kinase deficient (TKxe2x88x92) mutants can replicate in the periphery in dividing cells, but are replication-defective in trigeminal ganglion neurons (Coen et al., Proc. Natl. Acad. Sci. USA 86:4736-4740 (1989); Efstathiou et al., J. Gen. Virol. 70:869-879 (1989)). TKxe2x88x92 viruses may preferentially enter latency in sensory neurons (Kosz-Vnenchak et al., J. Virol. 64:5396-5402 (1990)), are unable to reactivate from latency to produce a lytic infection (Coen et al., Proc. Natl. Acad. Sci. USA 86:4736-4740 (1989); Efstathiou et al., J. Gen. Virol. 70:869-879 (1989)).
Several factors appear to limit the potential uses of herpes viruses for gene transfer to cells in culture and in vivo. These include the relatively frequent occurrence of spontaneous revertants of some mutants; possible recombination events between helper virus, viral sequences in plasmids, and latent sequences; deleterious effects on host cell macromolecular synthesis due to the presence of viral proteins; alterations in the host cell genome; and possible reactivation of preexisting latent viruses. (Davison et al., J. Gen. Virol. 65:859 (1984); Fenwick, M., Compr. Virol. 19:359 (1984); Heilbronn et al., J. Virol. 63:3683 (1989); Kwong et al., J. Virol. 63:4834 (1989); Johnson et al., 14th International Herpes Workshop abst. (1989)).
In the study of gene expression in neuronal cells in the nervous system, and the identification of therapies for treating neuronal disease of the nervous system, a need therefore continues to exist for methods of gene delivery with efficient viral vectors capable of mediating gene transfer into such cells without causing harm to the host animal.
The present invention is directed to a method for introducing gene sequences into cells of the nervous system, for example, in the cells of the central and peripheral nervous system using HSV-1 mutants as vectors for gene delivery. The HSV-1 virus is mutated so that it has a deletion(s) in a gene(s) necessary for viral replication in neurons, for example in sensory ganglia in vivo. The desired gene sequence to be delivered to the cell is inserted into the mutated HSV-1 such that the gene sequence will be expressed in the host cell. By the method according to this invention, a gene sequence is expressed in a cell in the nervous system by introducing the mutated HSV-1 vector with a mutation in a gene necessary for viral replication in neurons and with a gene sequence located downstream of a promoter sequence so that the gene sequence will be expressed in the host cell.
The vectors of the present invention are capable of stable expression of the foreign gene(s) in cells of the nervous system. Furthermore, by utilizing tissue or cell specific promoters, selective expression of foreign gene(s) can be maintained in cells of interest. Also, promoters could be utilized which are drug-inducible.
The invention also concerns a method for treating a neurological deficiency of the central nervous system by expressing a gene sequence, which complements the deficiency, in a cell in the central nervous system by introducing a HSV-1 vector with a mutation in a gene for viral replication and with a gene sequence downstream of a promoter sequence so that the gene sequence will be expressed in the cell and the expressed gene product complements the deficiency.
The invention is further directed to methods for modulating neuronal physiology by delivery of neuropeptide genes, such as those genes that express proteins or polypeptides to block pain including, for example, analgesic neuropeptides; genes that express growth factors including, for example, nerve growth factor; genes that express proteins or polypeptides to promote regeneration or prolong the life-span of a cell including, for example, superoxide dismutase; genes that express toxic proteins or polypeptides including, for example, ricin A, diphtheria toxin, and tetanus toxin, for example to kill tumor cells or to cause a cell-specific injury.
In the methods for modulating neuronal physiology, the method comprises introducing into a cell a HSV-1 vector with a mutation in a gene for viral replication and with a gene sequence coding for the desired protein or polypeptide located downstream of a promoter sequence so that the gene sequence will be expressed in the cell and upon expression will modulate the neuronal physiology.
The invention further concerns a method for treating persistent pain associated with nerve injury by expressing an analgesic neuropeptide gene sequence in a sensory neuron or in central nervous system neurons, by introducing a HSV-1 vector with a mutation in a gene for viral replication and with a gene sequence downstream of a promoter sequence so that the gene sequence is expressed in the cell and the expressed gene product substantially eliminates pain. In addition, vectors capable of causing a cell specific injury can also be used to relieve pain.
The present invention also relates to an animal model for painful neuropathy and a method for producing such an animal model. The animal model is produced by expressing a toxic gene sequence in a sensory neuron, wherein the expressed gene product causes cellular injury which injury results in painful neuropathy.
Enkephalin containing or other opioid peptide gene vectors can be introduced into regions of the central nervous system to induce analgesia (for example, periaquaductal grey). In addition, vectors capable of causing a cell specific injury can also be used to relieve pain in the present animal models.
The present invention further relates to an animal model for nervous system disease.
The methods and vectors of the invention can also be used to insert antisense sequences or ribozymes to reduce the expression of an endogenous protein associated with toxicity.